In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. Today, there exists a wide market of methods in which industry can accomplish these goals, one of which is chromatography. Chromatography is quite well suited to a variety of uses in the field of biotechnology, since it can separate complex mixtures with great precision and also is suitable for more delicate products, such as proteins, since the conditions under which it is performed are not typically severe.
One chromatography method, which is an especially sensitive separation technique and also applicable to most types of proteins, is metal chelate affinity chromatography (MCAC), also known as immobilised metal ion adsorption chromatography (IMAC). This technique is commonly used in purification schemes together with another chromatographic step, such ion exchange chromatography (IEX) and/or hydrophobic interaction chromatography (HIC).
More specifically, IMAC utilises matrices that comprises a group capable of forming a chelate with a transition metal ion, which chelate in turn is used as the ligand in chromatography to adsorb a compound from a liquid. The binding strength in IMAC is affected predominately by the species of metal ion, the pH of the buffers and the nature of the ligand used. Since the metal ions are strongly bound to the matrix, the adsorbed protein can be eluted either by lowering the pH or by competitive elution.
In general, IMAC is useful for separation of proteins or other molecules that present an affinity for the transition metal ion of the matrix. For example, proteins will bind to the matrix upon the presence of accessible histidine, cysteine and tryptophan residues, which all exhibit an affinity for the chelated metal.
With the advent of molecular biological techniques, proteins are now easily tailored or tagged with one or more histidine residues in order to increase their affinity to metal chelated ligands, and accordingly, metal chelate chromatography has more recently assumed a more important role in the purification of proteins.
Simple chelators have been suggested as ligands for IMAC, such as iminodiacetic acid (IDA). IDA, coupled to agarose supports and subsequent charged with various metals, such as Cu2+, Zn2+ and Ni2+, has been used for capture of proteins and peptides and is also available as commercial resins. More specifically, U.S. Pat. No. 4,551,271 (Hochuli, assigned to Hoffmann-La Roche Inc.) discloses a metal chelate resin which comprises IDA ligands, in the purification of interferon. The resin can be defined by the following formula:[agarose]-O—(CH2)—CHOH—CH2—N(CH2COO−)2Me2+,wherein Me is Ni or Cu.
The best results are obtained with this resin if the interferon has already been partially purified. The resin can according to the specification be prepared in a known manner by treating agarose with epichlorohydrin or epibromohydrin, reacting the resulting epoxide with iminoacetic acid disodium salt and converting the product into the copper or zinc salt by washing with a copper (II) or zinc solution.
More recently, EP 87109892.7 (F. Hoffmann-La Roche AG) and its equivalent U.S. Pat. No. 4,877,830 (Döbeli et al, assigned to Hoffmann-La Roche Inc.) disclosed a tetradentate chelator known as nitrilotriacetic acid (NTA) for use with metals that have six coordination sites. More specifically, the matrices can be described by the general formula:[carrier matrix]-spacer-NH—(CH2)x—CH(COOH)—N(CH2COO−)2Ni2+,wherein x=2–4. The disclosed matrix is prepared by reacting an amino acid compound of the formula R—HN—(CH2)x—CH(NH2)—COOH, wherein R is an amino protecting group and x is 2, 3 or 4, with bromoacetic acid in alkaline medium and subsequently, after an intermediate purification step, cleaving off the protecting group and reacting this group with an activated matrix. Accordingly, the method of preparation involves separate steps for alkylating and deprotecting the amino acid, which steps renders the method time-consuming and hence costly. In addition, the alkylation chemistry is less efficient, and after deprotection, the product is not well defined regarding rest products from neutralisation and cleavage. Following this, the material is coupled to a solid support that carries carboxyl functionalities by forming an amide bond. However, this procedure may involve disadvantages, since the media obtained presents the immobilised desired chelating ligand as well as some unreacted carboxylic groups, thus yielding a heterogeneous media. Furthermore, mono-N-protected amino acid compounds are expensive starting materials, rendering the overall method even more costly.
Finally, WO 01/81365 (Sigma-Aldrich Co.) discloses a metal chelating composition that according to the specification is capable of forming relatively stable chelates with metal ions and exhibits an improved selectivity for polyhistidine tagged proteins. According to said WO 01/81365, the linkage between the chelator and the resin is an important parameter for the selectivity, and the linkage is a neutral ether, a thioether, a selenoether or an amide. The disclosed compositions are coupled to an insoluble carrier, such as Sepharose™ according to given examples. The chromatographic media is produced in two different ways; either by a solid phase reaction directly on to the pre-activated solid support eventually used in the chromatographic media, or by a separate in solution synthesis of the intermediate product N,N,N′,N′-tetrakis (carboxymethyl)-L-cystine that is eventually coupled to the solid support.
The solid phase synthesis is carried out by adding L-cysteine to a previously epichlorhydrine activated Sepharose gel under alkaline conditions for a prolonged reaction time (18 h), followed by washings. Thereafter bromoacetic acid is added, again under alkaline conditions and a prolonged reaction time (72 h), and again followed by washings, and eventually capping of remaining free amino groups present on the gel with acetic acid anhydride.
Solid phase synthesis in this way offers poor control of the reaction and potential side reactions, and thereby yields a less homogeneous product.
The alternative route, relying on in solution phase synthesis of an intermediate product starts with addition of a large excess (40 times) of glyoxylic acid to L-cystine in an alkaline borate buffer. The intermediate product was thereafter, after pH manipulations and conductivity adjustment of the reaction mixture, purified with ion exchange chromatography to give N,N,N′,N′-tetrakis (carboxymethyl)-L-cystine.
Before coupling to a solid support the N,N,N′,N′-tetrakis (carboxymethyl)-L-cystine has to be reduced to N,N-bis(carboxymethyl)-L-cysteine using tris (carboxyethyl) phosphine under alkaline conditions. This material can finally be used for coupling to a pre-activated solid support forming the chromatographic media. This synthetic method is elaborate and depends on a large excess of reagents to form the desired product that is eventually purified under specific chromatographic conditions, followed by reduction as an additional synthetic step, and is thereby less suited for use in large-scale production.
Accordingly, there is still a need of improved methods for synthesis of IMAC ligands as well as of methods for the immobilisation thereof to a base matrix.